Sample preparation is an important part of many biological methods and is exceedingly important in situations where the biological sample is uncharacterized. In such situations there is a need for a universal method for extracting nucleic acids from a variety of bacterial cell and spores.
Sample preparation is also an important part of biotechnology, including microarray (biochip, microchip) technology. DNA microarray protocols require random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Sequence-independent fragmentation is necessary to reduce the size of the nucleic acids and minimize three-dimensional structure in the target region, making the target more accessible for on-chip hybridization. Fragmentation also allows different regions of a target molecule to independently interact with immobilized oligonucleotide probes.
Recently a Standard Protocol (S-Protocol) was developed for a single-tube and minicolumn format of nucleic acid isolation, fragmentation and labeling (U.S. Pat. No. 6,818,398 B2). The S-Protocol is based on radical-generating coordinating complexes chemistry (U.S. Pat. No. 7,208,269 B2), which is hereby incorporated by reference in its entirety. The S-Protocol was also configured for manual operation in the field (i.e. as a syringe-operated column), mobile, or stationary laboratory environment, and the extraction and analysis of nucleic acids from Gram positive and Gram negative vegetative bacteria. The protocol was validated for both DNA and RNA targets and was published (Bavykin et al., 2001, Appl. Environm. Microbiol. 67, 922-928; Kelly et al., 2002, Analyt. Biochem. 311, 103-118). One drawback to S-Protocol is that the lysis conditions are ineffective on spores or other recalcitrant organisms.
Several protocols have developed for extracting nucleic acids from spores and other recalcitrant organisms, however, these prior art techniques are inadequate for high yield of nucleic acids from such organisms because they focus on spore disruption techniques that only partially degrade or crack the outer membranes of the organism's complex system of cellular walls (i.e. spore). Many of these prior art methods tend to focus on physical disruption techniques such as sonication, glass bead mixing etc., heat shock treatment, taking advantage of germination (spore sprouting) and/or combinations thereof to disrupt the spore wall structure and release nucleic acids, followed by known extraction techniques to extract the released nucleic acids. As a result, many prior art techniques tend to release only extracellular nucleic acids that often exists in the spore samples as a result of incomplete spore purification from parental cells and fail to achieve high nucleic acid yields, which require extraction of intracellular nucleic acids. Furthermore, prior art protocols focus almost entirely on extraction from spores and are not appropriate for extraction of multiple cell types. While prior art methods may produce enough nucleic acid yield for PCR, such method do not typically produce adequate amounts of nucleic acid for direct analysis.
One method for extracting nucleic acids from spores is described by Moeller et al., “A Method for Extracting RNA from Dormant and Germinating Bacillus subtilis Strain 168 Endospores,” Current Microbiology Vol. 53, (2006), pp 227-231, herein referred to as “Moeller.” Moeller examined the extraction of nucleic acids from coated and decoated spores by germination (incubation times between of up to 120 minutes) followed by a acid-phenol extraction method. Therefore, Moeller's method actually represents protocol of nucleic acids extraction from vegetative cells, not from spores. Several prior art extraction methods also employ a germination/extraction approach including: Luna et al., “Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs, Journal of Clinical Microbiology, March 2003, 1252-1255, which combines sonication, autoclaving and germination to extract nucleic acids from spores. However, all of these methods may not be considered as genuine methods of nucleic acids isolation from dormant spores. In all of these methods dormant spores were converted in growing cells before the beginning of nucleic acids isolation.
Kuske et al., “Small Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil,” Applied and Environmental Microbiology, July 1998, 2463-2472, describes another typical spore extraction method combining the use of heat treatment, freeze-thaw cycles, and bead mill homogenization. While the Kuske method provides limited amounts of nucleic acids the yield is low which is typical of most prior art methods. Furthermore, the harsh conditions used by Kuske are not acceptable for most vegetative cells. See, also Van Assche et al., “The Pattern of Protein and Nucleic Acid Synthesis in Germinating Spores of Phycomyces blakesleeanus,” Arch. Mikrobiol. 93, 129-136 (1973), which combines heat shock with chemical disruption; Belgrader et al., “A Minisonicator to Rapidly Disrupt Bacterial Spores for DNA analysis,” Anal. Chem. 1999, 71, 4232-4236, which combines sonication and germination to achieve spore disruption; and Chandler et al., “Continuous Spore Disruption Using Rapidly Focused, High-Frequency Ultrasound,” Anal Chem. 2001, 73, 3784-3789, which employs high-frequency ultrasonication to achieve spore disruption.
Sargent et al., “A Procedure for Isolating High Quality DNA from Spores of Bacillus subtilis 168,” Journal of General Microbiology (1980), 116, 511-514, describes a complex method for isolating DNA from Bacillus spores. While the Sargent procedure produces a good yield (80%) it is very time-intensive (˜16 hours), is not suitable for use on a column due to the use of urea (and/or phenol), which tend to clog silica columns, and like most other spore extraction techniques is not suitable for use in mixture of multiple cell types like mixtures vegetative bacterial cells and bacterial spores. Other similar methods are disclosed by Papaphilis et al., “Defined conditions for DNA Extraction from Bacillus subtilis Spores,” Biochiim, Biohpys. Acta, 199 (1970) 548-550; and Dose et al., “DNA Stability and Survival of Bacillus Subtilis Spores in Extreme Dryness.
Given the limitation of prior art methods there exists a need for an effective, time-efficient method for extracting nucleic acids from a complex biological sample which is applicable for mixture of multiple types of cell including but not limited to: prokaryotic or eukaryotic cells and spores. The method would preferably be an on column method which could be easily automated and/or used in the field. Such a method would be especially useful for extracting nucleic acids from uncharacterized samples.